RNA in situ hybridization

Abstract

A method for identifying the presence of a target gene mRNA is provided, which involves hybridizing one or more oligonucleic acid probes with the target gene mRNA expressed in a tissue sample, and detecting a low-molecular-weight compound label added to at least one of the bases of the oligonucleic acid probes. The oligonucleic acid probes are contacted with the tissue sample for hybridization with the target gene mRNA after pretreating (prehybridizing) one or more dummy oligonucleic acids with the tissue sample, or a mixture of the oligonucleic acid probes and the dummy oligonucleic acids is contacted with the sample tissue to hybridize the oligonucleic acid probes with the target gene mRNA.

The invention claimed is: 
     
       1. An RNA in situ hybridization method for identifying a presence of an mRNA of a target gene in a tissue sample consisting of the steps of:
 (i) hybridizing the tissue sample with one or more oligonucleic acid probes labeled with a low-molecular-weight compound on at least one of the bases of the one or more oligonucleic acid probes, which hybridizes to the mRNA of the target gene expressed in the tissue sample and one or more different dummy oligonucleic acids that are substantially equal in length with the one or more oligonucleic acid probes, wherein the one or more different dummy oligonucleic acids neither hybridize with the mRNA of the target gene expressed in the tissue sample nor with the oligonucleic acid probes, and wherein the one or more dummy oligonucleic acids are adsorbed to sites of the tissue sample where the one or more of the oligonucleic acid probes are non-specifically adsorbed and prevent non-specific adsorption of one or more of the oligonucleic acid probes to the tissue sample and 
 (ii) detecting the low-molecular-weight compound with an antibody enzyme conjugate that binds to the low-molecular-weight compound and amplifying a signal using a color-developing compound or a fluorescent molecule compound as a substrate for the enzyme, and further detecting the signal by a 10× to 40× objective lens; thereby identifying the presence of the mRNA of the target gene. 
 
     
     
       2. The RNA in situ hybridization method of  claim 1 , wherein the amounts of the dummy oligonucleic acids are 2 to 10 times the amounts of the oligonucleic acid probes. 
     
     
       3. The RNA in situ hybridization method of  claim 2 , wherein the oligonucleic acid probes and the dummy oligonucleic acids are substantially equal in base length within a range of from 20 bp to 70 bp. 
     
     
       4. The RNA in situ hybridization method of  claim 1 , wherein the low-molecular-weight compound label is added to a 5′ end base and/or a 3′ end base of the oligonucleic acid probes. 
     
     
       5. The RNA in situ hybridization method of  claim 1 , wherein two or more of the oligonucleic acid probes are hybridized with the mRNA by being separated from each other by a distance of 8 or more bases between the 5′ end of one probe and the 3′ end of the other probe, wherein the low-molecular-weight compound label is added to a 5′ end base and/or a 3′ end base of the oligonucleic acid probes. 
     
     
       6. The RNA in situ hybridization method of  claim 1 , wherein the tissue sample is a tissue isolated from mammal, and wherein the dummy oligonucleic acids are oligonucleic acids that correspond to partial sequences of retrotransposon repeat sequences. 
     
     
       7. The RNA in situ hybridization method of  claim 1 , wherein the tissue sample is a tissue isolated from mammal, and wherein the dummy oligonucleic acids are oligonucleic acids that correspond to part of a plant genome, or partial sequences of a microorganism genome. 
     
     
       8. The RNA in situ hybridization method of  claim 1 , wherein the dummy oligonucleic acids are oligonucleic acids obtained by the A-to-T, T-to-A, G-to-C, and C-to-G substitutions of the base sequences of the oligonucleic acid probes,
 the dummy oligonucleic acids including the substitution of M×0.2 bases (rounded up to the nearest integer) to M×0.8 bases (rounded down to the nearest integer) with the complementary bases in a contiguous sequence of M or more same bases (M=4) when M or more same bases are present, and 
 the dummy oligonucleic acids including the substitution of at least N×0.2 bases (rounded up to the nearest integer) with the complementary bases in the presence of a palindromic sequence of N or more bases (N=5) identical to its complementary sequence when read from the 5′ side or 3′ side, the at least N×0.2 bases being at most (N/2−1) bases when N is an even number, and being at most ((N−1)/2−1) bases when N is an odd number. 
 
     
     
       9. A set of dummy oligonucleic acids used for the RNA in situ hybridization method of  claim 6 , wherein the dummy oligonucleic acids are partial sequences of retrotransposon repeat sequences, or different partial sequences of the repeat sequences. 
     
     
       10. A set of dummy oligonucleic acids for the RNA in situ hybridization method of  claim 7 , wherein the dummy oligonucleic acids are part of a plant genome or partial sequences of a microorganism genome, or different partial sequences of a plant genome or a microorganism genome.